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Abstract:

Localized delivery of 1,25 D3 directly to a target area using
biodegradable polymeric matrices maximizes the efficacy of this drug
while minimizing systemic exposure and toxicity. Anticalcemic analogs of
1,25 D3 have also been incorporated into controlled release polymer
formulations to achieve efficacious intracranial concentrations of 1,25
D3 analogs for the treatment of intracranial tumors as well as
neurodegenerative disorders such as Alzheimer's disease as well as to
maximize the efficacy of these analogs in the treatment of systemic
malignancies. The therapeutic efficacy of these formulations was
demonstrated through a variety of studies in vitro and in vivo. Hybrid
analogs of 1,25 D3 were incorporated into biodegradable polymer
wafers composed of a polyanhydride copolymer of
1,3-bis(p-carboxyphenoxy)propane (CPP) and sebacic acid (SA) in a 20:80
molar ratio. In addition to providing improved treatments for
malignancies and neurodegenerative disorders, the spatial localization
and high reproducibility of this controlled delivery methodology presents
a unique opportunity to study in vivo the poorly understood mechanisms of
1,25 D3's antiangiogenic, antiproliferative, and transcriptional
regulating activities.

Claims:

1. A controlled or sustained release formulation comprising vitamin D3 or
an analog thereof having antiproliferative activity, and a polymeric
matrix.

2. The formulation of claim 1 wherein the vitamin D3 or analog is present
in a dosage effective to inhibit proliferation or to cause toxicity of
malignant cells.

3. The formulation of claim 1 wherein the vitamin D3 or analog is present
in a dosage effective to induce expression of nerve growth factor.

4. The formulation of claim 1 wherein the formulation comprises a vitamin
D3 analog in a polymeric matrix.

5. The formulation of claim 4 wherein the vitamin D3 analog has the
formulawherein R1 is --OH or CH20H, R2 is a C4-6 chain or a C4-6
alkoxy chain, wherein the chain includes one or more substituents
selected from the group consisting of hydroxyl groups, preferably
tertiary hydroxyl groups, alkene groups, alkyne groups, alkyl groups,
preferably methyl and ethyl, and ketones, and R3 and R4 are either H or
together form a double bond.

6. The formulation of claim 4 wherein the analog has less calcemic
activity than vitamin D3.

7. The formulation of claim 5 wherein the analog is selected from the
group consisting of

[0003]The role of the seco-steroid hormone 1,25-Dihydroxyvitamin D3
(1,25 D3) in the regulation of calcium homeostasis and bone
metabolism via action in the intestine, bone, kidney, and parathyroid
glands has long been known. Recently, however, as the understanding of
the endocrinological impact of 1,25 D3 endocrinological impact has
broadened, a variety of new potentially therapeutic roles have emerged.
These include the treatment of a wide variety of neoplastic diseases, as
well as neurodegenerative disorders of the central nervous system (CNS).

[0005]Due to its demonstrated ability to upregulate Nerve Growth Factor
(NGF), a neurotrophic factor crucial to the maintenance of proper
cholinergic nerve function in the basal forebrain, hippocampus, and
cortex, 1,25 D3 has also been implicated in the treatment of
Alzheimer's disease. However, due to its limited penetration of the blood
brain barrier (BBB) and toxic systemic hypercalcemic effects, attempts to
upregulate in the brain by delivering 1,25D3 systemically have been
unsuccessful (Saporito, et al. Experimental Neurology, 123; 295-302,
1993). To bypass the BBB and reveal the therapeutic potential of
1,25D3 in the treatment of Alzheimer's, mini-osmotic pumps have been
utilized to deliver the drug into the murine brain
intracerebroventricularly (i.c.v.). (Carswell, S. Vitamin D in the
Nervous System: Actions and Therapeutic Potential. Vitamin 1: 1197-1211,
1997; Saporito, et al. Brain Research, 633; 189-196, 1994) Although no
NGF mRNA upregulation was observed following a single injection of
1,25D3 into the brain, pump-mediated chronic delivery for 6 days
resulted in pharmacologically relevant upregulation of NGF in cholinergic
neurons. The success of this treatment, however, was limited since i.c.v.
administration also results in high systemic concentrations of 1,25
D3 leading to dose-limiting toxic hypercalcemia. Furthermore, the
clinical application of this pump-mediated delivery system is perturbed
by a high incidence of infection and blockage of the catheter system.

[0006]To date, the most successful strategy for enhancing the therapeutic
index of 1,25 D3 has been the design and synthesis of unnatural
structural analogs with the objective of separating undesirable
calcitropic activity from potentially therapeutic anti-angiogenic,
antiproliferative, and transcriptional regulating activities (Elstner, et
al. Cancer. Res. 55:2822-2830 (1995); Zhou and Norman Endocrinology, 36;
1145-1152 (1995)). Several hundred 1,25 D3 analogs have been
prepared and tested worldwide, some of which appear successful in
achieving this goal in pre-clinical studies and are currently undergoing
small-scale clinical evaluation in the United States. The Posner group at
Johns Hopkins University has developed a methodology for separating 1,25
D3's desired and undesired activities which invokes the coupling of
various powerful antiproliferative enhancing structural units on the
C,D-ring side chain with an anticalcemic 1-b-hydroxymethyl A-ring
modification (Posner, et al. J. Org. Chem., 62: 3299-3314, 1997; Posner,
et al. J. Med. Chem., 35: 3280, 1992; Posner, et al. Bioorganic Medicinal
Chemistry Letters, 4: 2919, 1994). This strategy has yielded promising
new hybrid analogs that demonstrate retained antiproliferative activity
in vitro and dramatically minimized calcemic effects in vivo relative to
1,25 D3.

[0007]It is an object of this invention to provide vitamin D3 formulations
for treatment of cancer with reduced toxicity.

[0008]It is a further object of this invention to provide vitamin D3
formulations useful in treatment of neurodegenerative disorders.

SUMMARY OF THE INVENTION

[0009]Localized delivery of 1,25 D3 directly to a target area using
biodegradable polymeric matrices maximizes the efficacy of this drug
while minimizing systemic exposure and toxicity. Anticalcemic analogs of
1,25 D3 have also been incorporated into controlled release polymer
formulations to achieve efficacious intracranial concentrations of 1,25
D3 analogs for the treatment of intracranial tumors as well as
neurodegenerative disorders such as Alzheimer's disease as well as to
maximize the efficacy of these analogs in the treatment of systemic
malignancies. In addition to providing improved treatments for
malignancies and neurodegenerative disorders, the spatial localization
and high reproducibility of this controlled delivery methodology presents
a unique opportunity to study in vivo the poorly understood mechanisms of
1,25 D3's antiangiogenic, antiproliferative, and transcriptional
regulating activities.

[0010]The therapeutic efficacy of these formulations was demonstrated
through a variety of studies in vitro and in vivo. Hybrid analogs of 1,25
D3 were incorporated into biodegradable polymer wafers composed of a
polyanhydride copolymer of 1,3-bis(p-carboxyphenoxy)propane (CPP) and
sebacic acid (SA) in a 20:80 molar ratio. Various drug/polymer
combinations were co-dissolved in an organic solvent followed by drying
in vacuo. The resulting homogenous drug/polymer formulation was then
compression molded into cylindrical wafers using a miniature custom made
compression molding device, similar to micro KBr dies available from
Aldrich. Following systemic or intracranial implantation of drug loaded
polymer wafers, surface erosion of the polymer matrix over a period of
two to three weeks led to sustained release of these novel therapeutic
agents to a specific site within the body.

[0011]The results demonstrate that these drugs are potent inhibitors of
proliferation against a variety of murine tumor cell lines in vitro.
Strengthening the rationale for sustained drug delivery, a proportional
relationship between antiproliferative activity and exposure time was
shown. Evidencing therapeutic potential in the treatment of
neurodegenerative disorders such as Alzheimer's disease, studies
demonstrated that the 1,25 D3 analog MCW-YB can significantly
upregulate the synthesis of NGF by murine L929 fibroblasts in vitro. The
two most potent 1,25 D3 analogs demonstrate dramatically reduced
calcemic activity when compared to the parent compound. The most potent
hybrid analogs were also successfully loaded into biodegradable
polyanhydride copolymer wafers, and the sustained release of these
compounds from polymer wafers was demonstrated in vivo. These 1,25
D3 analog-loaded polymer wafers were well tolerated in the murine
brain and flank at drug loading doses ranging from 0.1 to 1% by weight.
Intracranial implantation of 5 mg pCPP:SA (20:80) polymer wafers loaded
with the 1,25 D3 analog JK-1626-2 or MCW-YB at 0.1% by weight
resulted in no significant weight loss or rises in blood ionized calcium
levels for 7 days. Similar implantation of 0.5% MCW-YB-loaded wafers into
Sprague-Dawley rats yielded no weight loss or rise in serum ionized
calcium for up to 12 days. Furthermore, the site-specific polymeric
delivery of 1,25 D3 analogs to the brain results in diminished
systemic hypercalcemia when compared to polymeric delivery to the flank.
Collectively, these studies reveal that sustained delivery via
biodegradable polymers of 1,25 D3 hybrid analogs are useful for the
treatment for several types of systemic and CNS malignancies, as well as
neurodegenerative disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a graph of the antiproliferative activity of 1,25 D3
and hybrid analogs at concentrations of 1, 10, 100, and 1000 nM against
murine B16 malignant melanoma cells. Results are expressed as % of
control, the mean cell number from 6 wells for each drug concentration
divided by the mean cell number from 6 control wells receiving only
solvent (isopropanol).

[0013]FIG. 2 is a graph of the antiproliferative activity of 1,25 D3
and hybrid analogs at 1, 10, 100 and 1000 nM against murine EMT6 breast
carcinoma cells. Results are expressed as % of control, the mean cell
number from 6 wells for each drug concentration divided by the mean cell
number from 6 control wells receiving only solvent (isopropanol).

[0014]FIG. 3 is a graph of the antiproliferative activity of 1,25 D3
and hybrid analogs at 1, 10, 100 and 1000 nM against murine RENCA renal
cell carcinoma cells. Results are expressed as % OF CONTROL, the mean
cell number from 6 wells for each drug concentration divided by the mean
cell number from 6 control wells receiving only solvent (isopropanol).

[0015]FIG. 4 is a graph of the exposure time dependent antiproliferative
activity of 1,25 D3 at 10 μM against B16 malignant melanoma
cells. Results are expressed as % of control, the mean cell number from 3
wells for each drug concentration divided by the mean cell number from 3
control wells receiving only solvent (0.4% isopropanol).

DETAILED DESCRIPTION OF THE INVENTION

[0016]Polymer-mediated delivery of 1,25 D3 or analogs thereof
directly to an intracranial target has several advantages including
circumvention of the blood brain barrier (BBB), achievement of high drug
concentrations in a desired locus, sustained drug delivery for up to five
years, and minimal systemic exposure and toxicity. Systemic application
of this polymer-based delivery strategy also offers the advantage of
maintaining constant, high levels of drug in a peripheral target area
with a smaller overall dose. The combination of controlled release
polymer formulations with analogs of 1,25 D3 characterized by low
calcemic activity and maintained therapeutic activities provides
additional advantages for treatment with both systemic and neurological
malignancies as well as neurodegenerative disorders such as Alzheimer's
disease.

I. Compositions

[0017]Vitamin D3 and D3 Analogs

[0018]D3 Analogs having anti-proliferative activity can be delivered using
controlled and/or sustained release formulations for treatment of cancer.
These have the following general and specific formulas and are described
by Posner, et al. J. Org. Chem., 62: 3299-3314, 1997; Posner, et al. J.
Med. Chem., 35: 3280, 1992; Posner, et al. Bioorganic Medicinal Chemistry
Letters, 4: 2919, 1994, the contents of which are hereby incorporated by
reference.

wherein R1 is --OH or CH2--OH, R2 is a C4-6 chain or a C4-6
alkoxy chain, wherein the chain includes one or more substituents
selected from the group consisting of hydroxyl groups, preferably
tertiary hydroxyl groups, alkene groups, alkyne groups, alkyl groups,
preferably methyl and ethyl, and ketones, and R3 and R4 are either H or
together form a double bond. The formula is also intended to include
fluorinated derivatives, with fluorines at one or more of the positions
shown in U.S. Pat. Nos. 5,428,029, 5,612,328, 5,039,671, and 5,451,574,
the contents of which are hereby incorporated by reference.

[0021]The Vitamin D3 derivatives are administered in controlled and/or
sustained release formulations. These can further include a
pharmaceutically acceptable carrier such as saline, phosphate buffered
saline, cells transduced with a gene encoding other bioactive molecules,
microparticles, or other conventional vehicles.

[0022]i. Polymeric Formulations

[0023]The Vitamin D3 derivatives can be encapsulated into a biocompatible
polymeric matrix, most preferably biodegradable. The Vitamin D3
derivative are preferably released by diffusion and/or degradation over a
therapeutically effective time, for example, between eight hours to five
years, more typically between one week and one year, depending on the
indication. As used herein, microencapsulated includes incorporated onto
or into or on microspheres, microparticles, or microcapsules.
Microcapsules is used interchangeably with microspheres and
microparticles, although it is understood that those skilled in the art
of encapsulation will recognize the differences in formulation methods,
release characteristics, and composition between these various
modalities. The microspheres can be directly implanted or delivered in a
physiologically compatible solution such as saline.

[0024]Biocompatible polymers can be categorized as biodegradable and
non-biodegradable. Biodegradable polymers degrade in vivo as a function
of chemical composition, method of manufacture, and implant structure.
Synthetic and natural polymers can be used although synthetic polymers
may be preferred due to more uniform and reproducible degradation and
other physical properties. Examples of synthetic polymers include
polyanhydrides, polyhydroxyacids such as polylactic acid, polyglycolic
acid and copolymers thereof, polyesters, polyamides, polyorthoesters, and
some polyphosphazenes. Examples of naturally occurring polymers include
proteins and polysaccharides such as collagen, hyaluronic acid, albumin
and gelatin. The ideal polymer must be processible and flexible enough so
that it does not crumble or fragment during use.

[0025]Vitamin D3 derivatives and optionally, other drugs or additives, can
be encapsulated within, throughout, and/or on the surface of the implant.
The Vitamin D3 derivative is released by diffusion, degradation of the
polymer, or a combination thereof. There are two general classes of
biodegradable polymers: those degrading by bulk erosion and those
degrading by surface erosion. The latter polymers are preferred where
more linear release is required. The time of release can be manipulated
by altering chemical composition; for example, by increasing the amount
of an aromatic monomer such as p-carboxyphenoxy propane (CPP) which is
copolymerized with a monomer such as sebacic acid (SA). A particularly
preferred polymer is CPP-SA (20:80). Use of polyanhydrides in controlled
delivery devices has been reported by Leong, et al., J. Med. Biomed.
Mater. Res., 19:941 (1985); J. Med. Biomed. Mater. Res., 20:51 (1986);
and Rosen, et al., Biomaterials, 4:131 (1983). U.S. patents that describe
the use of polyanhydrides for controlled delivery of substances include
U.S. Pat. No. 4,857,311 to Domb and Langer, U.S. Pat. No. 4,888,176 to
Langer, et al., and U.S. Pat. No. 4,789,724 to Domb and Langer. Other
polymers such as polylactic acid, polyglycolic acid, and copolymers
thereof have been commercially available as suture materials for a number
of years and can be readily formed into devices for drug delivery.

[0026]Non-biodegradable polymers remain intact in vivo for extended
periods of time (years). Agents loaded into the non-biodegradable polymer
matrix are released by diffusion through the polymer's micropore lattice
in a sustained and predictable fashion, which can be tailored to provide
a rapid or a slower release rate by altering the percent Vitamin D3
derivative loading, porosity of the matrix, and implant structure.
Ethylene-vinyl acetate copolymer (EVAc) is an example of a
nonbiodegradable polymer that has been used as a local delivery system
for proteins and other macromolecules, as reported by Langer, R., and
Folkman, J., Nature (London), 263:797-799 (1976). Others include
polyurethanes, polyacrylonitriles, and some polyphosphazenes.

[0027]In the preferred embodiment, only polymer and Vitamin D3 derivatives
to be released are incorporated into the delivery device, although other
biocompatible, preferably biodegradable or metabolizable, materials can
be included for processing purposes as well as additional therapeutic
agents.

[0028]Although not the preferred embodiment, polymeric gel formulations
can also be used to administer the drug. Many suitable polymeric
materials are known, including polyoxyethylene block copolymers such as
the Pluronics® and Poloxamers® marketed by BASF, photopolymerizable
gels such as those described by U.S. Pat. No. 5,573,934 to Hubbell, et
al.

[0029]ii. Additives

[0030]Buffers, acids and bases can be used to adjust the pH of the
composition. Agents to increase the diffusion distance of agents released
from the implanted polymer can also be included.

[0031]Fillers are water soluble or insoluble materials incorporated into
the formulation to add bulk. Types of fillers include sugars, starches
and celluloses. The amount of filler in the formulation will typically be
in the range of between about 1 and about 90% by weight.

[0032]Spheronization enhancers facilitate the production of spherical
implants. Substances such as zein, microcrystalline cellulose or
microcrystalline cellulose co-processed with sodium carboxymethyl
cellulose confer plasticity to the formulation as well as implant
strength and integrity. During spheronization, extrudates that are rigid,
but not plastic, result in the formation of dumbbell shaped implants
and/or a high proportion of fines. Extrudates that are plastic, but not
rigid, tend to agglomerate and form excessively large implants. A balance
between rigidity and plasticity must be maintained. The percent of
spheronization enhancer in a formulation depends on the other excipient
characteristics and is typically in the range of 10 to 90% (w/w).

[0033]Disintegrants are substances which, in the presence of liquid,
promote the disruption of the implants. The function of the disintegrant
is to counteract or neutralize the effect of any binding materials used
in the formulation. The mechanism of disintegration involves, in large
part, moisture absorption and swelling by an insoluble material. Examples
of disintegrants include croscarmellose sodium and crospovidone which are
typically incorporated into implants in the range of 1 to 20% of total
implant weight. In many cases, soluble fillers such as sugars (mannitol
and lactose) can also be added to facilitate disintegration of the
implants.

[0034]Surfactants may be necessary in implant formulations to enhance
wettability of poorly soluble or hydrophobic materials. Surfactants such
as polysorbates or sodium lauryl sulfate are, if necessary, used in low
concentrations, generally less than 5%.

[0035]Binders are adhesive materials that are incorporated in implant
formulations to bind powders and maintain implant integrity. Binders may
be added as dry powder or as solution. Sugars and natural and synthetic
polymers may act as binders. Materials added specifically as binders are
generally included in the range of about 0.5 to 15% w/w of the implant
formulation. Certain materials, such as microcrystalline cellulose, also
used as a spheronization enhancer, also have additional binding
properties.

[0036]Various coatings can be applied to modify the properties of the
implants. Three types of coatings are seal, gloss and enteric. The seal
coat prevents excess moisture uptake by the implants during the
application of aqueous based enteric coatings. The gloss coat improves
the handling of the finished product. Water-soluble materials such as
hydroxypropyl cellulose can be used to seal coat and gloss coat implants.
The seal coat and gloss coat are generally sprayed onto the implants
until an increase in weight between about 0.5% and about 5% preferably
about 1% for seal coat and about 3% for a gloss coat, has been obtained.

[0037]Enteric coatings consist of polymers which are insoluble in the low
pH (less than 3.0) of the stomach, but are soluble in the elevated pH
(greater than 4.0) of the small intestine. Polymers such as
Eudragit®, RohmTech, Inc., Malden, Mass., and Aquateric®, FMC
Corp., Philadelphia, Pa., can be used and are layered as thin membranes
onto the implants from aqueous solution or suspension. The enteric coat
is generally sprayed to a weight increase of about one to about 30%,
preferably about 10 to about 15%, and can contain coating adjuvants such
as plasticizers, surfactants, separating agents that reduce the tackiness
of the implants during coating, and coating permeability adjusters. Other
types of coatings having various dissolution or erosion properties can be
used to further modify implant behavior. Such coatings are readily known
to one of ordinary skill in the art.

[0038]iii. Manufacture of Controlled Release Devices

[0039]Controlled release devices are typically prepared in one of several
ways. The polymer can be melted, mixed with the substance to be
delivered, and then solidified by cooling. Such melt fabrication
processes require polymers having a melting point that is below the
temperature at which the substance to be delivered and polymer degrade or
become reactive. Alternatively, the device can be prepared by solvent
casting, where the polymer is dissolved in a solvent, and the substance
to be delivered is dissolved or dispersed in the polymer solution. The
solvent is then evaporated, leaving the substance in the polymeric
matrix. Solvent casting requires that the polymer be soluble in organic
solvents and that the agents to be encapsulated be soluble or dispersible
in the solvent. Similar devices can be made by solvent removal, phase
separation or emulsification or even spray drying techniques. In still
other methods, a powder of the polymer is mixed with the Vitamin D3
derivative and then compressed to form an implant.

[0040]Methods of producing implants also include granulation, extrusion,
and spheronization. A dry powder blend is produced including the desired
excipients and microspheres. The dry powder is granulated with water or
other non-solvents for microspheres such as oils and passed through an
extruder forming "strings" or "fibers" of wet massed material as it
passes through the extruder screen. The extrudate strings are placed in a
spheronizer which forms spherical particles by breakage of the strings
and repeated contact between the particles, the spheronizer walls and the
rotating spheronizer base plate. The implants are dried and screened to
remove aggregates and fines. These methods can be used to make
micro-implants (microparticles, microspheres, and microcapsules
encapsulating Vitamin D3 derivatives to be released), slabs or sheets,
films, tubes, and other structures.

II. Methods of Treatment

[0041]In the preferred embodiment the formulations are administered in a
tumor or other sites to be treated, most preferentially intracranially.
The dosage and formulation will be determined by the disorder to be
treated. More or less of the polymeric material, or the polymer loading,
can be used to treat the patient.

[0042]1,25 D3 analogs can also be administered in combination with
other chemotherapeutic agents such as cisplatin, BCNU, taxol, or
cytokines such as IL-2 to potentiate the effects of locally delivered
cytotoxic agents against solid tumors, alone or in combination with other
types of local or targeted or systemic therapy such as radiation. Drug
combinations for the treatment of neurodegenerative disorders can also be
used.

[0043]The spatial localization and high reproducibility of this controlled
delivery methodology also allows the study in vivo of the poorly
understood mechanisms of 1,25 D3's antiangiogenic,
antiproliferative, and transcriptional regulating activities.

EXAMPLES

[0044]The present invention will be further understood by reference to the
following non-limiting examples.

Example 1

Testing the Antiproliferative Activity of 1,25 D3 Hybrid Analogs
Against a Series of Murine Malignant Cell Lines in Vitro

[0045]Concentration Dependence in Proliferation Assays

[0046]In vitro proliferation assays were performed to measure the activity
of 1,25 D3 and its analogs against four murine metastatic tumor cell
lines, B16 (malignant melanoma), RENCA (renal cell carcinoma), EMT6
(breast cell carcinoma), CT26 (colon carcinoma). All cell lines were
grown and propagated in RPMI medium at 37° C. in 5% CO2.
Cultured cells were trypsinized and plated in triplicate at 10,000
cells/well in Falcon 24 well tissue culture plates. After 24 hours of
incubation the cells received fresh media containing either solvent
(isopropanol) or drug at concentrations ranging from 1-1000 nM (i.e., 1,
10, 100 or 1000 nM). When control wells neared confluence, cell number
was determined for each well as an average of two readings on a ZM
Coulter Counter Results are expressed as the average cell number for each
drug treatment group divided by the average cell number for the drug free
control group (designated as % OF CONTROL) vs. the concentration of drug
or analog.

[0047]The results are shown in FIGS. 1-3 and summarized in Table 1. Five
hybrid analogs, JK-III-7-2, MCW-068-Y-EE, JK-132-2, MCW-005-Y-B, and
JK-1626-2, and 1,25 D3 demonstrated significant antiproliferative
activity at 10 nM against B16 and RENCA (p<0.03), at 100 nM against
EMT6 (p<0.01), and at 1000 nM against CT26 (p<0.01, data not shown)
(JK-1626-2 not yet tested against RENCA and CT26). MCW-005-YB and
JK-1626-2 appeared to be the most potent analogs, consistently
demonstrating antiproliferative activity similar to that of the parent
compound.

[0048]Table 1 shows the antiproliferative effects of 1,25 D3 and four
hybrid analogs against B16 (malignant melanoma), RENCA (renal cell
carcinoma), and EMT6 (breast cell carcinoma). The concentration of each
drug required to effect 50% inhibition of cell proliferation, designated
as EC50, has been derived from the graphs shown in FIG. 2. The EC50 value
relative to that of 1,25 D3 has also been calculated to allow for
comparisons of drug potency.

[0049]Time Dependence Studies

[0050]In a series of exposure time dependence studies, B16 melanoma cells
were trypsinized, suspended, and plated as before. After 24 hours of
incubation original medium was removed and replaced with fresh medium
containing either solvent or drug at a concentration of 10 nM in
triplicate. Then at 1, 2, 10, 24, and 96 hours, the drug containing media
was removed and replaced with fresh media containing only solvent. Then
at 1, 2, 10, 24, and 96 hours the drug containing media was removed and
replaced with fresh media containing only solvent. At the 96 hour time
point, all groups were trypsinized and cell number was determined as
before.

[0051]FIG. 4 demonstrates the exposure time dependent antiproliferative
activity of 1,25 D3 at 10 μM against B16 malignant melanoma cells.
Results are expressed as % of control, the mean cell number from 3 wells
for each drug concentration divided by the mean cell number from 3
control wells receiving only solvent (0.4% isopropanol). These results
demonstrate that the antiproliferative activity of 1,25 D3 and its
analogs is exposure time dependent, strengthening the rationale for
sustained drug delivery as compared to bolus administration.

Example 2

Testing the Transcriptional Upregulation of NGF by 1,25 D3 and Hybrid
Analog MCW-YB in Murine L929 Fibroblasts In Vitro

[0052]In vitro studies were carried out to test the ability of 1,25
D3 and the analog MCW-YB to upregulate the expression of NGF in
murine L929 fibroblasts. L929 cells, obtained from ATCC (Rockville, Md.),
were harvested from culture and plated at 50,000 cells per well on a
Falcon 24 well tissue culture plate. After 24 hours of incubation,
culture media was removed from each well and replaced with serum free
medium containing either 1,25 D3 or MCW-YB at 100 nM or vehicle in
triplicate. After 48 hours of incubation, the media from each well was
quantitatively analyzed for NGF protein content using an enzyme linked
immunosorbant assay (ELISA). The total NGF production per 50,000 cells
was then determined using cell number values determined using a ZM
Coulter Counter as before.

[0053]Treatment with the analog MCW-YB led to statistically significant
(p<0.03) 40% increase in NGF expression compared to solvent controls.
It is important to note that similar small but significant increases in
NGF have been previously shown to be effective in the treatment of murine
models of Alzheimer's disease.

Example 3

Testing the Calcemic Activity of 1,25 D3 and the Two Most Potent
Hybrid Analogs, MCW-YB and JK-1626-2, in C57 BI/6 Mice

[0054]Having established that the Posner analogs of 1,25 D3
maintained their antiproliferative and transcriptional regulating
activities in vitro, it was determined whether the most potent analogs
MCW-YB and JK-1626-2 demonstrate substantially minimized calcemic
activity in vivo. To test for calcemic activity, 1,25 D3, MCW-YB,
and JK-1626-2 were dissolved in a biocompatible solvent composed of 80%
propylene glycol/20% phosphate buffered saline. Twenty-seven C57/B16 mice
(n=3 per group), received daily intraperitoneal injections solution
containing one of the three drugs at on of the following doses: 1, 10, or
100 mg/kg/day (corresponding to 0.02, 0.2, or 2 mg/day respectively).
Nine animals received daily intraperitoneal injections of solvent only to
serve as control. Animal weights were monitored daily at the time of
injection. On day 7, all animals were sacrificed and blood was collected
via cardiac puncture and quantitatively analyzed for ionized calcium
content at the Critical Care Lab at Johns Hopkins Hospital.

[0055]Treatment with the parent compound at 1 and 10 mg/kg/day led to
substantial toxic hypercalcemia, signified by substantial weight loss and
dramatic rises in blood ionized calcium levels. The group receiving 1,25
D3 at 100 mg/kg/day was so severely compromised that collection of
sufficient blood samples for ionized calcium quantification was not
possible. The hybrid analogs, however, were markedly less calcemic than
the parent compound. Remarkably, absolutely no signs of toxic
hypercalcemia were observed for the analog MCW-YB, i.e. no weight loss or
significant rise in blood ionized calcium, at the 1, 10 and even the 100
mg/kg/day dosing regimens. No weight loss was observed following
treatment with JK-1626-2 at 1 and 10 mg/kg/day as well. A small increase
in blood ionized calcium was observed at the 10 mg/kg/day dosing regimen,
but this was much less than the increase recorded for the parent compound
at the same dose. Significant weight loss and a rise in blood ionized
calcium were observed by day seven for the group receiving JK-1626-2 at
100 mg/kg/day, however both were significantly less severe than that
observed for 1,25 D3 at a 10× lower dose.

Example 4

Incorporation of 1,25 D3, MCW-YB, and JK-1626-2 into Biodegradable
Polyanhydride Polymer Wafers and Demonstration of Controlled Drug Release
In Vitro

[0058]To prepare the drug/polymer formulations, polymer and drug (various
% by weight loading) were co-dissolved in HPLC grade methylene chloride
and the solution was dried overnight in vacuo. The resulting homogenous
polymer formulation was compression molded into cylindrical wafers using
a miniature custom made compression molding device similar to micro KBr
dies available from Aldrich. This yielded 5 and 10 mg cylinders measuring
1.5 and 3 mm in diameter respectively and 0.5 mm in height. The polymer
wafers were stored in anhydrous conditions for later use.

[0059]In Vitro Release Studies.

[0060]To determine the release kinetics of MCW-YB and JK-1626-2 from the
pCPP:SA polymer formulations, 5 mg wafers were placed into 2 ml cryoware
cryogenic mini-vials. To each vial was added 2 ml of a 30% ethanol/70%
0.01M phosphate buffered aqueous solution (pH 7.4). The ethanol was added
to increase the solubility of the hydrophobic 1,25 D3 analogs. Vials
were incubated at 37° C. on an orbital shaker turning at 100 rpm.
Periodically the buffer solution was removed and replaced with fresh
buffer to approximate perfect sink conditions. The collected samples were
analyzed for 1,25 D3 analog content using quantitative high pressure
liquid chromatography (HPLC) with a Beckmann system Gold (including an
Autosampler 507, Programmable Solvent Module 126AA, and Programmable
Detector Module 166 from Beckmann Instruments, San Roman, Calif.)
controlled by Dell System 200 personal computer (Dell Computer
Corporation, Austin, Tx.) and equipped with 4.6×250 mm Microsorb-MV
C18 column (Rainin Instrument Company, Woburn, Mass.). The mobile phase
consisted of acetonitrile/water (60:40), the flow rates were 1.8
(MCW-YB), and 2.25 (JK-1626-2) ml/min. UV detection was performed at
wavelengths of 264 (MCW-YB) and 262 (JK1626-2) nM. Under these conditions
the retention time was 9.6 min. for MCW-YB and 17.1 min. for JK-1626-2.

[0061]Continuous drug release (50.2% total) was demonstrated in vitro over
a period of 110 hours for wafers loaded with MCW-YB at 2.1% (w/w). A
series of polymers loaded with JK-1626-2 at loading doses ranging from 1
to 10% demonstrated continuous release for up to 200 hours. These results
indicate that 1,25 D3 analogs can be loaded into pCPP:SA (20:80)
polymer formulations and released with maintained structural integrity in
vitro. However, in the absence of ethanol, drug release will most likely
occur more slowly, as would the case in vivo.

Example 5

Determining the Highest Tolerated Dose of MCW-YB and JK-1626-2 that can be
Delivered to the Murine Flank and/or Brain Via Biodegradable Polymer
Wafers

[0062]Determination of the Highest Tolerated Doses In Vivo

[0063]Using the hybrid analogs MCW-YB and JK-1626-2 loaded into pCPP:SA
(20:80) wafers, the highest tolerated dose of 1,25 D3 analogs that
could be polymerically delivered to the murine brain without systemic
toxicity due to hypercalcemia was determined. Polymer wafers with drug
loadings ranging from 0.01% to 1%, of each analog were prepared and
implanted in the brains of C57 B1/6 mice (n=4 per group). Animal weight
loss (an established indicator of hypercalcemia) were monitored daily.

[0064]The highest tolerated doses for JK-1626-2 and MCW-YB were 0.1% and
1% respectively. The dramatic increase in tolerance for MCW-YB correlates
well with the calcemic studies outlined in Example 3. Delivery of the
parent compound, 1,25 D3, to the brain of Sprague-Dawley rats using
a mini-osmotic pump implanted intracerebroventricularly (i.c.v.) resulted
in a rise in serum calcium after 6 days at the 60 ng/day dosing level. At
120 ng/day weight loss was observed, and reportedly at 240 ng/day the
animals were "severely compromised" by day 6. In contrast, 10 mg polymer
wafers loaded with 0.5% MCW-YB (50,000 ng of drug) implanted
intracranially in 9 Sprague-Dawley rats caused no weight loss in the
rats. Assuming a 20 day release period as is typical for the pCPP:SA
(20:80) wafers, these animals were receiving about 2500 ng of the 1,25
D3 analog MCW-YB per day (more than 10 times the dose of the parent
compound reported to have caused severe hypercalcemic toxicity when
delivered i.c.v.) and the study was carried out for twice as long (12
days). Analysis of blood samples collected via cardiac puncture at the
time of serial sacrifice on days 1, 6, and even 12 showed no significant
rise in blood calcium when compared to control animals receiving placebo
wafers.

Example 6

Testing the Hypothesis that Site-Specific Polymeric Delivery of 1,25
D3 Analogs can Result in Reduced Toxic Hypercalemia

[0065]The hypercalcemic toxicity of polymerically delivered MCW-YB and
JK-1626-2 was then compared to that of the parent compound, and used to
test the hypothesis that site-specific polymeric delivery of 1,25 D3
analogs can result in reduced toxic hypercalcemia. Twenty-four C57/B16
mice (n=3 per group) received intraflank or intracranial implantation of
5 mg pCPP:SA (20:80) polymer wafers loaded with no drug, 0.1% 1,25
D3, 0.1% MCW-YB, or 0.1% JK-1626-2. Animal weights were monitored
daily and blood was collected for quantitative ionized calcium analysis
via cardiac puncture on day 7 post-implantation.

[0066]Both intraflank and intracranial implantation of polymer wafers
loaded with 0.1% 1,25 D3 led to severe toxic hypercalcemia as
indicated by substantial weight loss and dramatic rises in blood ionized
calcium levels compared to placebo controls. In stark contrast, animals
treated with MCW-YB-loaded wafers showed no signs of toxic hypercalcemia
following implantation at either locus. Intracranial polymeric delivery
of the somewhat more calcemic analog, JK-1626-2, yielded no rise in blood
ionized calcium levels; however, a significant increase was observed in
animals receiving identical polymer wafers in the flank. This unique
result with JK-1626-2 demonstrates that indeed site-specific polymeric
delivery of 1,25 D3 analogs to the murine brain minimizes
hypercalcemic toxicity when compared to drug delivery to the flank.
Similar results would be expected with 1,25 D3 at a lower drug
loading dose and with MCW-YB at a higher dose.

Example 7

Testing the Efficacy of 1,25 D3 Analog-Loaded Polymer Wafers in the
Treatment of Malignancy In Vitro and In Vivo

[0067]In vitro proliferation assays in which the 1,25 D3 analogs were
delivered from drug-loaded pCPP:SA (20:80) wafers were used to evaluate
initially the therapeutic potential of 1,25 D3 analog-loaded polymer
wafers in the treatment of cancer. Cultured murine B16 malignant melanoma
cells were trypsinized and plated at 5000 cells/well in Falcon 6 well
tissue culture plates. After 24 hours to allow for cell attachment, 0.5
mg polymer wafers, created by sectioning a 5 mg wafer into 10 pieces,
loaded with various amounts of MCW-YB or JK-1626-2, were added to cell
culture media. Control wells received 0.5 mg placebo polymers. When
control wells neared confluence, all wells were harvested and cell number
was determined as before on a ZM Coulter Counter.

[0069]The therapeutic efficacy of this strategy was also tested in vivo. A
solid tumor flank model was developed in which 50,000 EMT6 breast
carcinoma cells harvested from culture are injected subcutaneously in
Balb-C mice; after nine days, palpable solid flank tumors are observed
(MCW-005-YB EMT6 Breast Carcinoma Model). In the first study using this
model, tumors were measured on day 9 and animals were randomized into two
treatment groups. Seven mice received placebo polymer wafers and 7 mice
received wafers loaded with. MCW-YB at half the highest tolerated
intracranial dose (0.5% w/w) in the flank. Tumor volume was measured
every other day in a blinded fashion using venier calipers and animal
weights were periodically determined.

[0070]The results indicate that MCW-YB, when delivered locally from
pCPP:SA wafers, inhibits the growth of EMT6 solid tumors. However, due to
low numbers of animals included in each group and unexpected lethal
toxicity observed in the treatment arm the results were not statistically
significant.

[0071]In conclusion, these studies demonstrate the therapeutic potential
of controlled release polymers loaded with anticalcemic analogs of 1,25
D3, in the treatment of a variety of malignancies as well
neurodegenerative disorders such as Alzheimer's disease.